PROJECT SUMMARY/ABSTRACT Alzheimer?s disease is one of a family of tauopathies, neurological disorders in which the microtubule- associated protein tau becomes abnormally regulated. Although heritable mutations in the tau gene (MAPT) cause a subset of familial tauopathies, it is not well understood how this genetic variation contributes to the deleterious effects of mutant tau in the brain. The Ashe lab has shown that tau with the P301L mutation, the most common MAPT mutation, mislocalizes within neurons from axons to dendritic spines, through a process involving proteolytic cleavage of tau at aspartate 314 (D314). The D314 tau cleavage product is associated with cognitive deficits in humans with Alzheimer?s, and inhibiting this cleavage event prevents neuronal mislocalization of P301L tau. Our preliminary data further show that, in six-week-old transgenic mice, cleavage of P301L tau leads to elevated tau levels in the brain, which can be reversed by rendering tau non-cleavable at D314. Multiple disease-linked MAPT mutations alter the global conformation of tau in a way similar to the P301L mutation, and can also cause subcellular mislocalization and elevation of tau protein levels. Therefore, we hypothesize that cleavage at D314 promotes missorting of tau harboring pathogenic mutations, and leads to elevated protein levels early in disease, an effect that can be reversed by inhibiting cleavage at D314. We will address this hypothesis with the following Specific Aims. Specific Aim 1 examines the effect of cleavage at D314 on the half-life and localization of P301L tau. Using precision transgenesis, we have developed novel mouse lines called the T-series, genetically equivalent in every way except for single mutations we have introduced into a tau transgene. In the brains of these mice engineered to express wild-type (WT) tau, P301L tau, and non-cleavable P301L tau, we will measure the in vivo half-life and subcellular localization of tau. Specific Aim 2 investigates the effect of cleavage at D314 on early protein levels of pathogenic mutant tau. We will generate T-series mice expressing cleavable or non-cleavable tau with known pathogenic mutations, including the G272V, V337M, and R406W mutations, and measure tau protein levels in the brain at six weeks. The experiments included in these aims will elucidate the relationship between localization and degradation of tau, and may identify a robust and early biomarker for preclinical studies of tauopathies, while also providing the field with novel mouse lines and vectors to further investigate tau biology in disease. Because there may be common mechanisms between familial and sporadic tauopathies, understanding the effects of MAPT mutations on the biochemical properties of tau may lead to more effective therapies for a broad range of incurable diseases.